The use of natural products, including proteins, is a well known method of controlling many insect, fungal, viral, bacterial, and nematode pathogens. For example, δ-endotoxin proteins of Bacillus thuringiensis (B.t.) are used to control both lepidopteran and coleopteran insect pests. Genes producing these proteins have been introduced into and expressed by various plants, including cotton, tobacco, corn, wheat, rice, potato, and tomato, a number of different varieties of forage and turf grasses, ornamental flowers, and other fruit and vegetable crops. There are, however, several economically important insect pests that are not particularly susceptible to B.t. endotoxins. Examples of such important pests are the boll weevil (BWV), Anthonomus grandis, and corn rootworm (CRW), Diabrotica spp. In addition, having other, different gene products which do not function like Bt proteins for control of insects which are susceptible to B.t. endotoxins is important, if not vital, for effective and long term resistance management practices.
Recently, alternative species of bacteria have been identified which are capable of producing proteins displaying insect inhibitory effects. Photorhabdus and Xenorhabdus comprise broad genus' of bacteria which occupy the gut of entomopathogenic nematodes. upon invasion of the insect body by the nematode, the entomopathogenic bacteria are released from the gut of the nematode into the insect haemolymph where they proliferate, inhibit further development of the insect, and produce a nutrient enriched monoculture designed specifically for symbiotic nematode and bacterial survival. A variety of extracellular proteins are produced by these bacterial symbionts, each insect inhibitory protein having distinct insect genus and species specificity, each protein likely being structurally and probably functionally different from BT ICP's. (Ensign et al., Insecticidal Protein Toxins from Photorhabdus, WO 97/17432; Jarrett et al., Pesticidal Agents, WO 98/08388; Ffrench-Constant et al., Novel insecticidal Toxins from Nematode-Symbiotic Bacteria, Cellular and Molecular Life Sciences 57:828–833, May 2000).
Plant proteins have also been identified which exhibit insect inhibitory effects. One such protein is patatin, a non-specific lipid acyl hydrolase, which is the major storage protein of potato tubers (Gaillaird, T., Biochem. J. 121: 379–390, 1971; Racusen, D., Can. J. Bot., 62: 1640–1644, 1984; Andrews, D. L., et al., Biochem. J., 252: 199–206, 1988). Patatin has been shown to control various insects, including western rootworm (WCRW, Diabrotica virigifera), southern corn rootworm (SCRW, Diabrotica undecimpunctata), and boll weevil (BWV, Anthonomus grandis) (U.S. Pat. No. 5,743,477, issued Apr. 28, 1998). Patatin related protein sequences have been identified in a variety of plant species. When applied at an appropriate level in artificial diet, potato patatin is lethal to some larvae and will stunt the growth of survivors so that maturation is prevented or severely delayed, resulting in no reproduction. These proteins display non-specific lipid acyl hydrolase activity. Studies have shown that the enzyme activity is essential for its insect inhibitory activity (Strickland, J. A., et al., Plant Physiol., 109: 667–674, 1995). Patatins may be applied directly to the plants or introduced in other ways well known in the art, such as through the application of plant-colonizing microorganisms, which have been transformed to produce the enzymes, or by the plants themselves after similar transformation.
In potato, the patatins are found predominantly in tubers, but also at much lower levels in other plant organs (Hofgen, R. and Willmitzer, L., Plant Science, 66: 221–230, 1990). Genes that encode patatins have been previously isolated by Mignery, G. A., et al. (Nucleic Acids Research, 12: 7987–8000, 1984; Mignery, G. A., et al., Gene, 62: 27–44, 1988; Stiekema, et al., Plant Mol. Biol., 11: 255–269, 1988) and others. Patatins are found in other plants, particularly solanaceous species (Ganal, et al., Mol. Gen. Genetics, 225: 501–509, 1991; Vancanneyt, et al., Plant Cell, 1: 533–540, 1989) and recently Zea mays (Patent number WO 96/37615). Rosahl, et al. (EMBO J., 6: 1155–1159, 1987) transferred a patatin coding sequence into tobacco plants, and observed expression of patatin, demonstrating that patatin can be heterologously expressed by plants. Modification of coding sequences has been demonstrated to improve expression of other insect inhibitory protein genes such as the δ-endotoxin sequences from Bacillus thuringiensis (Fischhoff and Perlak; WO 93/07278). However, expression of a native plant species sequence encoding a protein exhibiting insect inhibitory properties in a plant at levels not previously observed in nature would be particularly advantageous. Such sequences would not require coding sequence modifications found to be necessary to achieve substantial levels of insect protection as have been required for sequences encoding Bt proteins for example.
As indicated above, plant non-specific lipid acyl hydrolases have been identified from a variety of plant sources including potato tubers. Speculation on the role of the enzyme has been centered on their involvement in the turnover of membrane lipids, however one report identified an serine residue required for hydrolase activity and conserved sequence flanking the residue in potato patatin based on inactivation of the enzyme acyl lipid hydrolase activity when treated with diisopropyl fluorophosphate and an amino acid sequence alignment with a patatin isoform (Walsh et al., U.S. Pat. No. 5,743,477; Apr. 28, 1998). Based on the amino acid sequence of potato patatin, Walsh et al. proposed that Ser-77 in the hydrolase motif, Gly-X-Ser-X-Gly is the catalytic residue required for enzyme function as well as insect inhibitory activity.
The inventors herein have identified a patatin isozyme designated Pat17, and used alanine scanning mutagenesis and X-ray crystallography to solve the structure of the patatin enzyme and to identify additional residues responsible for both catalytic activity and insect inhibitory bioactivity.
Novel proteins generated by the method of sequence transposition resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al. Proc. Natl. Sci., U.S.A., 76: 3218–3222, 1979; Teather, et al., J. Bacteriol., 172: 3837–3841, 1990; Schimming, et al., Eur. J. Biochem., 204: 13–19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127–130, 1991; MacGregor, et al., FEBS. Lett., 378: 263–266, 1996). The first in vitro application of sequence rearrangement to proteins was described by Goldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165: 407–413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion or sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain. This approach has been applied to proteins which range in size from 58 to 462 amino acids and represent a broad range of structural classes (Goldenberg and Creighton, J. Mol. Biol., 165: 407–413, 1983; Li and Coffino, Mol. Cell. Biol., 13: 2377–2383, 1993; Zhang, et al., Nature Struct. Biol., 1: 434–438, 1995; Buchwalder, et al., Biochemistry, 31: 1621–1630, 1994; Protasova, et al., Prot. Eng., 7: 1373–1377, 1995; Mullins, et al., J. Am. Chem. Soc., 116: 5529–5533, 1994; Garrett, et al., Protein Science, 5: 204–211, 1996; Hahn, et al., Proc. Natl. Acad. Sci. U.S.A., 91: 10417–10421, 1994; Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980–11984, 1993; Luger, et al., Science, 243: 206–210, 1989; Luger, et al., Prot. Eng., 3: 249–258, 1990; Lin, et al., Protein Science, 4: 159–166, 1995; Vignais, et al., Protein Science, 4: 994–1000, 1995; Ritco-Vonsovici, et al., Biochemistry, 34: 16543–16551, 1995; Horlick, et al., Protein Eng., 5: 427–431, 1992; Kreitman, et al., Cytokine, 7: 311–318, 1995; Viguera, et al., Mol. Biol., 247: 670–681, 1995; Koebnik and Kramer, J. Mol. Biol., 250: 617–626, 1995; Kreitman, et al., Proc. Natl. Acad. Sci., 91: 6889–6893, 1994).
Thus, there exists a need to identify novel protein sequences which are insect inhibitory, which are not related to Bt insect inhibitory proteins in form or function, and which are safe for expression in human and animal food supplies. Such proteins should have modes of action distinct from those of Bt insect inhibitory proteins or Xenorhabdus or Photorhabdus insect inhibitory proteins and should act synergistically with BT's or Xenorhabdus or Photorhabdus insect inhibitory proteins to aid in preventing the onset of insect species resistance developed in response to providing only single insect inhibitory proteins in compositions of matter as food sources to populations of insects in fields of recombinant crops.